Fig. 4
HACE1 interacts with and attenuates ubiquitin degradation of NRF2. a, b Treatment of the HACE1 knockdown- or HACE1/HACE1C876S overexpression-SF295 cells and control cells with 10 μM cycloheximide at the time indicated. Left panels represent western blot analysis of the indicated proteins. The band intensity of NRF2 in cycloheximide-treated cells was normalized to that of GAPDH (loading control), and subsequently normalized to that in DMSO-treated cells (right panels). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01. c HACE1 knockdown-SF295 cells and control cells were pretreated with 25 µM MG132 or DMSO for 4 h, and western blot analysis was then carried out to assess protein expression of HACE1, NRF2, and GAPDH (loading control). d HACE1/HACE1C876S overexpression-SF295 cells and control cells were pretreated with 25 µM MG132 for 4 h. Next, cells were lysated and mixed with anti-NRF2 antibody conjugated with agarose. Immunoblotting was then carried out to assess NRF2 ubiquitination using anti-ubiquitin (Ub) antibody. Input samples were taken prior to the above steps. e SF295 cells stably expressing wild-type HACE1 or the indicated mutants and control cells were lysated and immunoprecipitated (IP) with anti-NRF2 antibody, and the precipitated proteins were subjected to western blot analysis using anti-KEAP1 antibody. f, g The interaction between HACE1 and NRF2 was evaluated by reciprocal Co-IP assays in HACE1/HACE1C876S overexpression-SF295 cells and control cells. h Immunofluorescence staining showing the co-localization of HACE1 and NRF2 in the extranuclear region of SF295 and U87 cells. Blue color represents DAPI staining for nuclei; Red color represents HACE1 tagged with red fluorescence protein (RFP); green color represents NRF2. i Immunofluorescence assay showing the co-localization of HACE1 and NRF2 in the endoplasmic reticulum of SF295 and U87 cells. Blue color represents DAPI staining for nuclei; Red color represents HACE1 tagged with RFP or NRF2; Green color represents calnexin. Scale bars, 20 μm
